Natural killer (NK) cells are large granular lymphocytes of bone marrow origin that exhibit cytolytic activity against certain tumor and virally infected cells in the absence of prior stimulation and without MHC- restriction. The molecular basis of NK cell recognition and activation by target cells is poorly understood. We recently described a cell surface molecule 2B4 that is expressed on all NK cells and T cell subsets that mediate non-MHC-restricted lysis. Ligation of the 2B4 molecule activates cytolysis and secretion IFNgamma by NK and non-MHC-restricted T cells. 2B4 belongs to the immunoglobulin superfamily and structurally resembles CD48 and LFA-3. In this proposal we will test the hypothesis that 2B4 is involved in the mediation of non-MHC-restricted lysis. To this end, we will create a soluble chimeric 2B4-Fc molecule and use it as a probe to identify the ligand for 2B4 on the surface of target cells. 2B4 ligand will be cloned from the appropriate cells by expression cloning. We will determine whether NK resistant target cells could be made susceptible to killing by expression of 2B4 ligand in target cells. A 2B4 genomic clone has been obtained and is being characterized so as to create mice null in 2B4 expression by homologous recombination. We will test NK cell functions in 2B4 knockout mouse by a variety of assays. The experiments proposed here will provide a better understanding of NK cell activation and recognition via 2B4. The long term objective is to devise strategies for eliminating tumor cells by selective expression of ligands in tumor cells.